Global gene expression profiling reveals similarities and differences among mouse pluripotent stem cells of different origins and strains

Pluripotent stem cell lines with similar phenotypes can be derived from both blastocysts (embryonic stem cells, ESC) and primordial germ cells (embryonic germ cells, EGC). Here, we present a compendium DNA microarray analysis of multiple mouse ESCs and EGCs from different genetic backgrounds (strains 129 and C57BL/6) cultured under standard conditions and in differentiation-promoting conditions by the withdrawal of Leukemia Inhibitory Factor (LIF) or treatment with retinoic acid (RA). All pluripotent cell lines showed similar gene expression patterns, which separated them clearly from other tissue stem cells with lower developmental potency. Differences between pluripotent lines derived from different sources (ESC vs. EGC) were smaller than differences between lines derived from different mouse strains (129 vs. C57BL/6). Even in the differentiation-promoting conditions, these pluripotent cells showed the same general trends of gene expression changes regardless of their origin and genetic background. These data indicate that ESCs and EGCs are indistinguishable based on global gene expression patterns alone. On the other hand, a detailed comparison between a group of ESC lines and a group of EGC lines identified 20 signature genes whose average expression levels were consistently higher in ESC lines, and 84 signature genes whose average expression levels were consistently higher in EGC lines, irrespective of mouse strains. Similar analysis identified 250 signature genes whose average expression levels were consistently higher in a group of 129 cell lines, and 337 signature genes whose average expression levels were consistently higher in a group of C57BL/6 cell lines. Although none of the genes was exclusively expressed in either ESCs versus EGCs or 129 versus C57BL/6, in combination these signature genes provide a reliable separation and identification of each cell type. Differentiation-promoting conditions also revealed some minor differences between the cell lines. For example, in the presence of RA, EGCs showed a lower expression of muscle- and cardiac-related genes and a higher expression of gonad-related genes than ESCs. Taken together, the results provide a rich source of information about the similarities and differences between ESCs and EGCs as well as 129 lines and C57BL/6 lines. Such information will be crucial to our understanding of pluripotent stem cells. The results also underscore the importance of studying multiple cell lines from different strains when making comparisons based on gene expression analysis.     

Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat embryonic germ cells

Pluripotent cells referred to as embryonic germ cells (EGCs) can be derived from the embryonic precursors of the mature gametes: the primordial germ cells (PGCs). A homozygous mutation (ter) of the dead-end homolog 1 gene (Dnd1) in the rat causes gonadal teratocarcinogenesis and sterility due to neoplastic transformation and loss of germ cells. We mated heterozygous ter/+ WKY-Dnd1^ter/Ztm rats and were able to cultivate the first genital ridge-derived EGCs of the rat embryo at day 14.5 post coitum (pc). Genotyping revealed that ten EGC lines were Dnd1 deficient, while only one wild type cell line had survived in culture. This suggests that the inactivation of the putative tumor suppressor gene Dnd1 facilitates the immortalization of late EGCs in vitro. Injection of the wild type EGCs into blastocysts resulted in the first germ-line competent chimeras. These new pluripotent rat EGCs offer an innovative approach for studies on germ cell tumor development as well as a new tool for genetic manipulations in rats.     

原始生殖细胞发生的机制

生殖细胞的发生是发育和遗传的基础。在几乎所有哺乳动物中,原始生殖细胞（primordial germ cell,PGC）均由近端上胚层体细胞在周边细胞特定的信号诱导下特化而成。目前的研究已经发现一些与生殖细胞特化有关的信号分子和关键转录调控元件,以及特化后生殖细胞获得的与体细胞不同的生物特性。生殖细胞的特化是一个结合了体细胞发育程序的抑制、细胞多能性程序的启动和全基因组表观遗传重编程三个方面的动态的复杂过程。多能性干细胞（胚胎干细胞或诱导型多能干细胞）具有发育全能性,能分化为机体任何一种细胞类型,包括生殖细胞。利用多能性干细胞体外分化形成生殖细胞有助于深入系统地研究配子发生的调控机制,为干细胞在不育症治疗方面的应用带来新希望。     

胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展

胚胎干细胞（embryonic stem cells,ESCs）具有自我更新、无限增殖和多向分化的特性,包括分化成心脏组织的多种类型细胞。经体细胞重编程产生的诱导多能干细胞（induced pluripotent stem cells,iPS）也被证明有类似胚胎干细胞的特性。但这些多能干细胞向心肌细胞自发分化的效率非常低,因此,如何有效地诱导这些多能干细胞向心肌细胞的定向分化对深入认识心肌发生发育的关键调控机制和实现其在药物发现和再生医学,如心肌梗塞、心力衰竭的细胞治疗以及心肌组织工程中的应用均具有非常重要的意义。该文重点综述了近年来胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展,并探讨了这一研究领域亟待解决的关键问题和这些多能干细胞的应用前景。     

Differentiation-promoting effects of mammary-derived growth inhibitor (MDGI) on pluripotent mouse embryonic stem cells

Pluripotent embryonic stem cells (line BLC6), when cultivated in vitro as embryoid bodies and injected subcutaneously into syngeneic mice, form teratocarcinomas consisting of embryonal carcinoma cells and differentiated tissues of all three primary germ layers. In order to study the possible effects of the mammary-derived growth inhibitor (MDGI) on the differentiation pattern of the tumors developing in the mice, BLC6 cell-derived embryoid bodies were treated in vitro for 4 days with either MDGI or a synthetic peptide composed of the C-terminal 11 amino acids of MDGI. In those tumors, significantly more differentiated neural tissue and lesser proportions of undifferentiated embryonic carcinoma cells (ECC) were found in the MDGI-and peptide-treated groups, compared with controls. The results are discussed with respect to a possible differentiation-promoting capacity of MDGI.     

Generation of rabbit pluripotent stem cell lines

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

Gene Expression Signatures of Extracellular Matrix and Growth Factors during Embryonic Stem Cell Differentiation

Pluripotent stem cells are uniquely capable of differentiating into somatic cell derivatives of all three germ lineages, therefore holding tremendous promise for developmental biology studies and regenerative medicine therapies. Although temporal patterns of phenotypic gene expression have been relatively well characterized during the course of differentiation, coincident patterns of endogenous extracellular matrix (ECM) and growth factor expression that accompany pluripotent stem cell differentiation remain much less well-defined. Thus, the objective of this study was to examine the global dynamic profiles of ECM and growth factor genes associated with early stages of pluripotent mouse embryonic stem cell (ESC) differentiation. Gene expression analysis of ECM and growth factors by ESCs differentiating as embryoid bodies for up to 14 days was assessed using PCR arrays (172 unique genes total), and the results were examined using a variety of data mining methods. As expected, decreases in the expression of genes regulating pluripotent stem cell fate preceded subsequent increases in morphogen expression associated with differentiation. Pathway analysis generated solely from ECM and growth factor gene expression highlighted morphogenic cell processes within the embryoid bodies, such as cell growth, migration, and intercellular signaling, that are required for primitive tissue and organ developmental events. In addition, systems analysis of ECM and growth factor gene expression alone identified intracellular molecules and signaling pathways involved in the progression of pluripotent stem cell differentiation that were not contained within the array data set. Overall, these studies represent a novel framework to dissect the complex, dynamic nature of the extracellular biochemical milieu of stem cell microenvironments that regulate pluripotent cell fate decisions and morphogenesis.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

牛胚胎生殖细胞的分离与培养

胚胎生殖细胞 (Embryonicgermcells,EG)是由生殖嵴原始生殖细胞 (Primordialgermcells,PGCs)中分离得到的一种未分化而多潜能的干细胞。牛EG细胞的研究在EG细胞核移植、转基因及建立生物反应器方面具有广阔的应用前景。本研究从 2 9- 70日龄牛胎儿PGCs分离得到EG细胞 ,经过抑制分化培养 ,其中一个细胞系传至 6代。所分离得到的EG细胞具有典型的EG细胞形态 ,AP及PAS染色呈阳性 ,核型正常 ,同时观察到这些细胞在体外进行自发性分化 ,可形成类胚体、成纤维样细胞及神经样细胞